We will determine the effects of epigenetic modifications on replication origin activation and silencing within the immunoglobulin heavy chain (Igh) locus. We have established the location of many replication initiation sites throughout the Igh locus and have determined how they change during B cell development. We will now focus on modifying the Igh locus in murine ES and proB cells to understand how chromatin modifications associated with active and inactive chromatin domains affect the activation of initiation domains of DNA replication within the locus. For these studies, we have engineered ES cells to contain an exchangeable cassette at two sites within the Igh locus. Using these ES cells, we propose to target to these sites, specific proteins that modify chromatin structure and have histone modifying activities. We already have demonstrated that we can tether a transcriptional activator to Gal4 target sites that we have inserted in the Igh region, resulting in histone modifications and the activation of a previously silent gene. We will also modify the locus by inserting much larger targeting sequences containing frequent recognition sites. We will determine the effects of epigenetic modifications on replication initiation, the direction and rate of replication fork progression and on the time in S phase during which the Igh and flanking regions replicate. We will use two novel approaches that we have recently developed: single molecule analysis of replicated DNA (SMARD) and high-resolution timing array (HRTA) analysis. The role of higher order chromosome structure in regulating replication will also be investigated. We will use the ES cells with the Igh locus, modified by cassette exchange, to produce chimeric mice and homozygous knock-in mice to determine the effect on B cell development of epigenetic changes in the Igh locus. We will characterize these changes using the detailed information we have already obtained about replication initiation and fork direction, and nuclear organization of the Igh locus at different stages of normal development in the B lineage.